Characterization of enzymes in the vitamin K cycle

项目总结/摘要 维生素K循环酶：γ-谷氨酰羧化酶（GGCX）、维生素K环氧化物还原酶（VKOR）和 维生素K还原酶（VKR）负责维生素K依赖性（VKD）的翻译后羧化 将蛋白质转化为生物活性形式。羧基化主要与血液凝固有关，因为四 凝血因子（因子II、VII、IX和X）和三种抗凝蛋白（蛋白质C、S和Z）需要 羧化作用。随着新的VKD蛋白及其新的生物学功能的发现， 羧化作用的重要性已扩展到血管钙化、骨发育、葡萄糖 代谢和细胞增殖。GGCX是直接修饰VKD蛋白以使其功能 forms. GGCX的遗传变异已在维生素K相关疾病患者中发现。但 目前尚不清楚为什么一些GGCX突变导致出血性疾病，而其他突变导致非出血性疾病 症状此外，一个GGCX突变有时会导致两种不同的临床表型。但 目前还不清楚为什么服用维生素K可以改善一种症状，而不能改善另一种症状。VKOR 是维生素K循环的调节酶，也是口服抗凝剂华法林的靶点。机制 VKOR的活性部位再生仍然难以捉摸最近的研究表明，超级华法林中毒 (an比华法林更有效抗凝剂）是一个日益增长的公共健康问题， 因为这既昂贵又低效。将维生素K还原为对苯二酚形式以支持VKD的酶 羧化是VKR。尽管经过几十年的努力，VKR的身份仍然未知。我们的长期研究目标 详细了解所有维生素K循环酶的结构和功能关系， 更好地控制凝血和其他相关生理过程的环境。目前的建议旨在 解决该领域仍然存在的主要问题（如上所述）。为了实现这些目标，我们建议 具体目标1 -我们将使用我们最近建立的CRISPR（定期更新） 间隔短回文重复序列）-Cas9敲除报告细胞系以研究所有目前的基因的作用。 鉴定了不同VKD蛋白羧化上天然存在的GGCX突变， 独特的临床表型;具体目标2 -我们将应用遗传密码扩展技术来阐明VKOR的 活性位点再生和设计合成新型维生素K衍生物以拯救超华法林 和特异性目标3 -我们将使用全基因组CRISPR-Cas9敲除文库来筛选 未知酶VKR我们希望从这些研究中获得的信息将有助于我们了解 三种维生素K循环酶有助于羧化的复杂机制，从而获得新的 控制血液凝固和血管钙化的治疗见解，并改善 维生素K相关疾病

Project Summary/Abstract The vitamin K cycle enzymes: gamma-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR) and vitamin K reductase (VKR) are responsible for the post-translational carboxylation of vitamin K-dependent (VKD) proteins into their biologically active forms. Carboxylation is mainly associated with blood coagulation, as four coagulation factors (factors II, VII, IX, and X) and three anticoagulant proteins (proteins C, S, and Z) require carboxylation for their function. With the discovery of new VKD proteins and their new biological functions, the importance of carboxylation has been expanded to vascular calcification, bone development, glucose metabolism, and cell proliferation. GGCX is the enzyme that directly modifies VKD proteins to their functional forms. Genetic variations in GGCX have been identified in patients with vitamin K-related disorders. However, it is not clear why some GGCX mutations cause bleeding disorders while other mutations result in non-bleeding symptoms. Additionally, one GGCX mutation can sometimes cause two distinct clinical phenotypes. However, it remains unclear as to why the administration of vitamin K can ameliorate one symptom but not the other. VKOR is a regulatory enzyme of the vitamin K cycle and the target of the oral anticoagulant, warfarin. The mechanism of VKOR's active site regeneration remains elusive. Recent studies have shown that superwarfarin poisonings (an anticoagulant more powerful than warfarin) are a growing public health concern and that vitamin K therapy for that is costly and inefficient. The enzyme that reduces vitamin K to its hydroquinone form to support VKD carboxylation is VKR. Despite decades of effort, the identity of VKR is still unknown. Our long-term research goal is to understand in detail the structure and function relationship of all vitamin K cycle enzymes within their native milieu for better control coagulation and other related physiological processes. The current proposal aims to solve the main questions remaining in the field (as mentioned above). To accomplish these goals, we propose the following specific aims: Specific Aim 1 - we will use our recently established CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 knockout reporter cell lines to study the effect of all currently identified naturally occurring GGCX mutations on the carboxylation of different VKD proteins associated with distinct clinical phenotypes; Specific Aim 2 - we will apply genetic code expansion technology to clarify VKOR's active site regeneration and design and synthesize novel vitamin K derivatives to rescue superwarfarin poisonings; and Specific Aim 3 - we will use the genome-wide CRISPR-Cas9 knockout library to screen for the unknown enzyme, VKR. We expect that information derived from these studies will help us understand how the three vitamin K cycle enzymes contribute to the complex mechanisms of carboxylation, thereby gaining new therapeutic insights into the control of blood coagulation and vascular calcification and improving therapies for vitamin K-related disorders.